Three New Species of Carcinonemertes (Nemertea, Carcinonemertidae) from the Southeastern Coast of Brazil

Journal of Natural History, 2006; 40(15-16): 915-930 (^) Taylor & Francis Taylor & Francis Group

Three new species of Carcinonemertes (Nemertea, Carcinonemertidae) from the southeastern coast of Brazil

CYNTHIA SANTOS1, JON L. NORENBURG1 & SERGIO L. S. BUENO2

1 Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institute, Washington, DC 20560-0163, USA, and 2Departamento de Zoologia, Instituto de Biociencias, Universidade de Sao Paulo, Rua do Matao, travessa 14, n° 101, 05508-900, Butantan, Sao Paulo, SP, Brazil

(Accepted 31 December 2005)

Abstract Three new species of Carcinonemertes from the southeastern coast of Brazil are described: Carcinonemertes divae new species, Carcinonemertes caissarum new species and Carcinonemertes sebastianensis new species. They were found, respectively, associated with the crabs Libinia spinosa, Hepatus pudibundus and Menippe nodifrons, each a newly recorded host for Carcinonemertes. Characters not previously used to describe members of the family Carcinonemertidae, such as distance from ovaries to tip of head, distance from brain to tip of head and distance from stylet to tip of head are included in the descriptions of the new species and are discussed. The locations of these new carcinonemertid worms in their respective hosts are presented in detail and a novelty regarding the infestation site is registered.

Keywords: Carcinonemertidae, decapod crustacean, symbiosis, predation, Carcinonemertes divae, Carcinonemertes caissarum, Carcinonemertes sebastianensis, Brazil

Introduction Nemertean worms of the family Carcinonemertidae are symbiotic egg predators of many decapod crustaceans. Owing to their life cycle, intimacy and use of chemically mediated cues from their hosts, their biology is effectively akin to parasitism; their ecological impact, however, is that of a predator because they kill individual embryos (Kuris 1993; Torchin et al. 1996). The family Carcinonemertidae comprises two genera, Carcinonemertes (Kolliker 1845), which lacks accessory stylets, and Ovicides Shields, 2001, the latter with a single species, O. juliae Shields, 2001, which has accessory stylets. Ten species of Carcinonemertes have been described so far: C. carcinophila (Kolliker 1845), with two recognized subspecies, C. c. carcinophila (Kolliker 1845) and C. c. imminuta Humes, 1942; C. epialti Coe ,1902; C.

Correspondence: Cynthia Santos, Department of Invertebrate Zoology, Smithsonian Institute, PO Box 37012, National Museum of Natural History W-216, MRC 163, Washington, DC 20013-7012, USA. Email: [email protected] Published 22 August 2006 ISSN 0022-2933 print/ISSN 1464-5262 online © 2006 Taylor & Francis DOI: 10.1080/00222930600833842 916 C. Santos et al. mitsukurii Takakura, 1910; C. coei Humes, 1942; C. errans Wickham, 1978; C. regicides Shields, Wickham & Kuris, 1989; C. australiensis Campbell, Gibson & Evans, 1989; C. humesi Gibson & Jones, 1990; C. wickhami Shields & Kuris, 1990, and C. pinnotheridophila McDermott & Gibson, 1993 (Shields 2001). To date only Carcinonemertes carcinophila imminuta has been reported for Brazil, in the portunid crabs Callinectes danae Smith and Callinectes ornatus Ordway (Santos and Bueno 2001). In the present paper, three new species of the genus Carcinonemertes are described, obtained from the crabs Libinia spinosa H. Milne Edwards (Decapoda, Pisidae), Hepatus pudibundus (Herbst) (Decapoda, Hepatidae) and Menippe nodifrons Stimpson (Decapoda, Menippidae) collected on the southeastern coast of Brazil. The locations of these new worms in their respective hosts are presented in detail. The distinctive morphological characters of the new species are compared to the previously described ones; new characters are included in the descriptions and discussed.

Material and methods Decapod crustaceans were collected by trawling on the southeastern coast of Brazil along the following beaches: Anchovas Beach, Sao Sebastiao Island, (23°55'S, 45°19'W); Enseada Beach, Sao Sebastiao, (23°43'S, 45°25'W), and Poco Beach, Sao Sebastiao Island, (23°45'S, 45°16'W). Crabs were also collected by hand at Figueira Beach, Sao Sebastiao (23°44'56"S, 45°24'34"W). Crabs were transported to the nearby laboratory facilities at the Centro de Biologia Marinha, Universidade de Sao Paulo, Sao Sebastiao, where they were kept alive in tanks with a flow-through seawater system until dissection. Identification of crabs followed Melo (1996). The exoskeleton surface and the arthrodial membranes of the crabs were macroscopically examined for nemerteans. The dorsal carapace was removed to expose the branchial chambers. Gills and pleopods were removed with the aid of forceps and were examined under a dissecting microscope. The nemerteans were collected from crabs and placed in Petri dishes filled with seawater until the moment of taking measurements, pictures and notes on characters. Nemerteans were relaxed in a 1:1 solution of 7.5% MgCl2 (prepared with bottled drinking water) and seawater for 15-30 min, after which, length and width of body were determined with the aid of an ocular micrometer in a dissecting microscope. Measurements of internal features were made with the aid of an ocular micrometer in a compound microscope after covering the worms with a coverslip. Photomicrographs were made with a Canon PowerShot A10 digital camera. Holotype and paratypes of each new species are deposited at the Museu de Zoologia da Universidade de Sao Paulo, Brazil (Abbreviation: MZUSP). Some mucus sheaths produced by Carcinonemertes sebastianensis n. sp were prepared for scanning electron microscopy to obtain more details on their morphology. The mucus sheaths were cleaned in a 1:1 solution of 7.5% MgC12 (prepared with bottled drinking water) and seawater and were fixed in a 10% seawater formalin solution. Mucus sheaths were dehydrated in a series of graded ethanol (50, 70, 95, 100 and 100% for 10 min each), dried by the critical point method with C02 and coated with gold, then examined with a JEOL 6400 Visions scanning electron microscope. New Carcinonemertes from Brazil 917

Systematics Family Carcinonemertidae Sumner, Osburn & Cole, 1913

Diagnosis Modified by Shields et al. (1989) from Humes (1942): Monostiliferous hoplonemerteans living as symbionts (egg predators) on the gills, under the abdomen, on the apodemes, and axillae, and in or on the egg masses of decapod crustaceans. Short proboscis, reaching scarcely beyond the posterior end of the muscular portion of the esophagus. Lateral nerves lie internal to the well-developed submuscular glands. Cephalic glands well developed, with cephalic muscle fibers present. Cerebral organs lacking. Takakura's duct system present in males. Internal fertilization and oviparity occur commonly. In most species, adult worms occupy, at least temporarily, mucus sheaths secreted and attached to the setae on the pleopods and hairs of endopodites of ovigerous decapods. Embryos hatch as hoplone- mertean larvae.

Genus Carcinonemertes (Kolliker, 1845)

Diagnosis From Coe (1902) and amended here (italic): Nemerteans living as symbionts (eggpredators) on various species of Crustacea. Proboscis but little developed, very small in size, and extremely short, without lateral pouches of reserve stylets, but armed with central stylet and basis only; anterior proboscis very short, without distinct muscular layers, without distinct nerves, and without a thickened glandular epithelium. Cerebral organs lacking. Two ocelli. Cephalic glands massively developed. Usually oviparous, though fertilization often takes place internally.

Carcinonemertes divae new species (Figure 1A-F)

Diagnosis Body color varies from translucent white to orange. Two eyes, black. Anterior end of body rounded or pointed; posterior end pointed. Worms 1.3-4.3 mm long. Accessory stylets absent. Ovaries arranged in one row on each side of intestine. Takakura's duct present. Ornamented and filiform mucus sheath attached to pleopods of hosts may be present in adult worms.

Material examined Fourteen females, 16 males and one larva were examined. Holotype: male, from the egg mass of Libinia spinosa; type locality: Poco Beach, Sao Sebastiao Island, Brazil, (23°45'S, 45°16'W), 18 Jul 2003; Coll. Cynthia Santos; MZUSP No. 001. Paratype: female, from the egg mass of L. spinosa; type locality: Poco Beach, Sao Sebastiao Island, Brazil (23°45'S, 45°16'W); 18 Jul 2003; Coll. Cynthia Santos; MZUSP No. 002. 918 C. Santos et al.

Figure 1. Carcinonemertes divae n. sp. (A) Anterior end of female, (B) proboscis aligned in a straight line, (C) stylet and basis (arrow) in a female worm, (D) male with rounded anterior end, (E) pointed posterior end of male, with seminal vesicle, (F) adult female (arrow) on Libinia spinosa eggs. Abbreviations: b, proboscis bulb; d, diaphragm; p, posterior proboscis chamber.

Etymology The species name is a noun in the genitive singular and honors Dr Diva D. Correa, a Brazilian nemertean specialist, formerly from the Universidade de Sao Paulo, who dedicated almost 40 years of her life to the study of nemerteans and greatly contributed to the knowledge of this phylum. New Carcinonemertes from Brazil 919

Description The description is based on living adults and one larva. The latter was obtained from crabs collected at Anchovas Beach. Measurements are given as mean+SE (range, number of specimens observed).

Female. Body color varied from translucent white to orange; gut orange; gonads translucent white. Two eyes, black, irregular, circular or elliptical; elliptical eyes the most common shape. Found free among egg mass of host or in a filiform, ornamented mucus sheath attached to the pleopods. Lapilli homogeneously distributed on mucus sheath and 4+1 urn (3-5 |im; n=2) in height. Anterior end of body rounded or pointed (Figure 1 A); posterior end pointed. Dimensions of relaxed worms 2.6 + 0.1 mm (1.3- 3.2mm; ra=14) long and 332+17um (200-480um; n=14) wide. Eye 26+1 urn (20- 36 um; w=13) long and 16 + 1 |im (10-20 um; w=13) wide. Distance between eyes 103 +4 um (80-130 um; «=12). Distance from eyes to tip of head 143 + 9.5 um (100- 204 um; w=12). Brain 117 + 9um (92-150um; n=l) long and 57 + 3um (45-70um; n=7) wide. Distance from brain to tip of head 171 +7um (140-200 um; w=8). Proboscis aligned in a straight line (Figure IB). Anterior proboscis chamber 75 um (w=l) long and 12um («=1) wide. Diaphragm 53 + 2 um (42-80 um; w=14) long and 54 + 3 um (45-78 um; M=14) wide. Single stylet on basis, 10+1 um (8-12 um; w=13) long. Stylet basis 25 + 1 um (22-30um; n=14) long and 7 + 0um (5-8um; w=14) wide (Figure 1C). Stylet:basis ratio 0.387 (0.250-0.500; w=13). Distance from central stylet to tip of head 22l+2Oum (130- 400 um; n= 12). Accessory stylets absent. Proboscis bulb 36 + 2 um (22-48 um; n= 12) long and 45 + 2 um (38-56 um; w=14) wide. Posterior proboscis 57 + 6 um (30-72 um; n=6) long and 51 + 5 um (35-68 um; w=7) wide. Ovaries arranged in one row on each side of the intestinal diverticula. Number of ovaries 31+4 (13-53; w=ll). Distance from first gonad to tip of head 428+17um (348-540 um; w=12).

Male. Body color cream; gut orange; gonads translucent white. Two eyes, black, irregular, circular or elliptical; elliptical eyes the most common shape. Found free among egg mass of host or in a filiform, ornamented mucus sheath attached to the pleopods. Lapilli homogeneously distributed on mucus sheath and 4um (w=l) in height. Anterior end of body rounded or pointed (Figure ID), posterior end pointed (Figure IE). Dimensions of relaxed worms 2.6 + 0.2 mm (1.3-4.3 mm; w=14) long and 316 + 32 um (170-720 um; w=16) wide. Eye 29 + 2 um (22-48 um; w=16) long and 17 + 1 um (10- 25 um; w=16) wide. Distance between eyes 96 + 3um (75-112 um; w=15). Distance from eyes to tip of head 160+10um (110-230um; w=16). Brain 118 + 9um (90-175um; M=9) long and 52 + 3um (45-70 um; w=7) wide. Distance from brain to tip of head 212 +5 um (200-260 um; w=13). Proboscis aligned in a straight line. Anterior proboscis chamber 46 + 6um (40-52 um; w=2) long and 18 um (w=2) wide. Diaphragm 48 + 1 um (45-55 um; w=14) long and 50 + 2um (38-60 um; w=14) wide. Single stylet on basis 10 + 0um (8-12um; w=13) long. Stylet basis 28 +1 um (22-30um; n=15) long and 7 + 0 um (5-10um; w=15) wide. Stylet:basis ratio 0.371 (0.250-0.500; w=13). Distance from central stylet to tip of head 231 +14 um (160-310 um; w=13). Accessory stylets absent. Proboscis bulb 35 + 2um (25-48 um; w=13) long and 39 + 2 um (25-48 um; M=13) wide. Posterior proboscis 72+11 um (40-100um; n=5) long and 48+1 um (25- 50 um; n=6) wide. Distance from first gonad to tip of head 475 +9 um (370-500 um; n=12). Seminal vesicle (Figure IE) easily visible under stereomicroscope in the majority of adult male worms. 920 C. Santos et al.

Larva. Body ciliated with anterior and posterior ciliary tuft. Body shape spherical or ovoid. Two eyes. Length 110(J.m (w=l). Width 70|im (w=l). Anterior tuft 20(J.m (w=l) in length. Posterior tuft 24 \im (n= 1) in length. Eye 12 (J.m (n= 1) long and 6 |0xn (n= 1) wide. Distance between eyes 22p,m (w=l). Distance from eyes to tip of head 33|im (w=l).

Infestation site Adult worms were found in the host's egg mass (Figure IF) (ovigerous females with eggs in initial, intermediate and final stages of development). Immature worms were found on the abdomen of juvenile male crabs; on the abdomen and at the arthrodial membrane of pereopods of juvenile female crabs; at the base of pereopods, on the ventral and dorsal sides of the abdomen of adult male crabs; on the ventral and dorsal sides of the abdomen ofnon- ovigerous adult female crabs; at the base of pleopods, on the abdomen and on the eggs of ovigerous females; at the base of pleopods and on the ventral side of the abdomen of post- ovigerous females.

Figure 2. Carcinonemertes caissarum n. sp. (A) Filiform mucus sheath of adult worm; (B) male with rounded posterior end; (C) stylet and basis (arrow) in a male worm; (D) juvenile worm encysted on pleopod setae of Hepatus pudibundus. New Carcinonemertes from Brazil 921

Carcinonemertes caissarum new species (Figure 2A-D)

Diagnosis Body color varies from translucent white to cream; male with a red spot at posterior end. Two eyes, black. Anterior end of body rounded, posterior end rounded or truncated (males). Worms 1.2-11.0mm long. Accessory stylets absent. Ovaries arranged in one row on each side of intestine. Takakura's duct present. Ornamented and filiform mucus sheath may be present in adult worms.

Material examined Nine females and eleven males were examined. Holotype: female, from the egg mass of Hepatus pudibundus; type locality: Poco Beach, Sao Sebastiao Island, Brazil, (23°45'S, 45°16'W), 16 September 2002; Coll. Cynthia Santos; MZUSP No. 003. Paratype: female, from the egg mass of H. pudibundus; type locality Poco Beach, Sao Sebastiao Island, Brazil, (23°45'S, 45°16'W), 16 September 2002; Coll. Cynthia Santos; MZUSP No. 004.

Etymology The species name is a noun in the genitive plural and is in recognition of the human communities known as "caigaras" (a tupi-guarani word), which are found in the localities where the crabs were collected. The people of these communities depend mainly on fisheries; therefore, this name honors the fishermen who helped us collect, Rogerio dos Santos Junior and his father, Rogerio dos Santos.

Description The description is based on living adults. Measurements are given in mean + SE (range, number of specimens observed).

Female. Body color varied from translucent white to cream; gut varied from orange to brownish; gonads translucent white. Two eyes, black, round or cup-shaped; round eyes the most common shape. Found free among egg mass of host or in a filiform, ornamented mucus sheath (Figure2A). Lapilli 7 + 0um (7-8 urn; n=2) in height, larger at center of sheath and smaller and scarce at extremities. Anterior and posterior end of body rounded. Dimensions of relaxed worms 5.5 +1.0mm (2.6-11.0mm; w=7) long and 282 + 20um (210-376 urn; w=8) wide. Eye ll+9um (8-15 um; «=9) long and 9 + lum (5-12 um; M=9) wide. Distance between eyes 112+11 um (75-160 um; w=7). Distance from eyes to tip of head 115+ 6 urn (100-155 um; n=9). Brain 97 + 6um (88-120 um; n=5) long and 46 + 2 um (40-52 um; n=l) wide. Distance from brain to tip of head 132 +6 um (120- 160 um; w=6). Anterior proboscis chamber could not be distinguished. Diaphragm 38 + 2um (30-48 um; w=8) long and 46 + 2 um (38-55 um; w=7) wide. Single stylet on basis 8+lum (5-10 um; w=8) long. Stylet basis 22+lum (20-25 um; w=9) long and 7 + 0um (5-8um; «=9) wide. Stylet:basis ratio 0.378 (0.250-0.500; w=8). Distance from central stylet to tip of head 160 + 8 um (122-190 um; w=8). Accessory stylets absent. Proboscis bulb 26 + 2 um (20-38 um; w=8) long and 30 + 2 um (22-38 um; w=8) wide. 922 C. Santos et al.

Posterior proboscis 70±4|j.m (55-88 um; «=4) long and 59±4|j.m (52-68 (J.m; «=4) wide. Ovaries arranged in one row on each side of the intestinal diverticula. Number of ovaries 158 + 30 (76-252; w=5). Distance from first gonad to tip of head 568 + 56|im (430-900 |im; w=8).

Male. Body color cream with a red spot at the posterior end; gut varied from orange to brownish; gonads translucent white. Two eyes, black, irregular, circular or cup-shaped. Found free among egg mass of host or in a filiform, ornamented mucus sheath. Lapilli 4 \xxn (w=l) in height. Anterior end of body rounded, posterior end rounded (Figure 2B) or truncated. Dimensions of relaxed worms 2.0 + 0.3 mm (1.2-4.0 mm; w=9) long and 400+ 25 urn (230-520 um; w=ll) wide. Eye 11 + lum (8-15 um; w=10) long and 10 + 0urn (8-12 um; w=10) wide. Distance between eyes 107 + 9um (62-150[im; w=8). Distance from eyes to tip of head 106 + 7um (60-130 um; w=10). Brain 110 + 6um (86- 125 |im; n=8) long and 54 + 4|im (38-75 urn; n=8) wide. Distance from brain to tip of head 135 +9 um (80-170 um; w=8). Anterior proboscis chamber could not be distinguished. Diaphragm 42 + 4 |im (30-60 \im; n=l) long and 42 + 2um (32-50 |im; M=7) wide. Single stylet on basis 8+1 |im (5-10 urn; w=9) long. Stylet basis 21 + 1 um (18- 25 um; w=10) long and 7 + 0|im (5-8|im; n=9) wide (Figure2C). Stylet:basis ratio 0.372 (0.250-0.571; n=9). Distance from central stylet to tip of head 172 + 13 um (110-216 um; w=8). Accessory stylets absent. Proboscis bulb 22 + 2 um (20-25 uni; n=2) long and 30 \im (w=l) wide. Posterior proboscis could not be observed. Distance from first gonad to tip of head 325 + 46um (230-510 um; n=5). Seminal vesicle easily visible under stereomicroscope in the majority of adult male worms. Infestation site Adult worms were found in the host's egg mass (ovigerous females with eggs in initial, intermediate and final stages of development). Immature worms were found on the ventral side of the abdomen of adult male crabs; on the abdomen, at the gonopores, at the base of pleopods and encysted on the setae of pleopods (Figure 2D) of non-ovigerous adult female crabs; encysted on the setae of pleopods of ovigerous females; on the abdomen, at the gonopores, on the arthrodial membrane of pleopods, and encysted on the setae of pleopods of post-ovigerous females.

Carcinonemertes sebastianensis new species (Figures 3, 4A-E and 5A, B)

Diagnosis Body color translucent white. Two eyes, brown. Anterior and posterior end of body rounded. Worms 2.0-11.0 mm long. Accessory stylets absent. Ovaries arranged in one row on each side of intestine. Distance from first ovary to tip of head 1250-2000 um. Takakura's duct present. Ornamented and filiform mucus sheath may be present in adult worms.

Material examined Four females, six males, 12 eggs and one larva were examined. Holotype: male, from the egg mass of Menippe nodifrons; type locality: Figueira Beach, Sao Sebastiao, Brazil, New Carcinonemertes from Brazil 923

Figure 3. Scanning electron micrograph of the lapilli of the mucus sheath produced by the adults of Carcinonemertes sebastianensis n. sp.

(23°44'56"S, 45°24'34"W); 22 January 2004; Coll. Cynthia Santos; MZUSP No. 005. Paratype: female, from the egg mass of M. nodifrons; type locality: Figueira Beach, Sao Sebastiao, Brazil, (23°44'56"S, 45°24'34"W); 22 January 2004; Coll. Cynthia Santos; MZUSP No. 006.

Etymology The species name is an adjective in feminine singular and is in recognition of the locality where the host crab was found, Sao Sebastiao.

Description The description is based on living adults, eggs and larva. Measurements are given in mean + SE (range, number of specimens observed).

Female. Body color translucent white; gut varied from orange to brownish; gonads translucent white. Two eyes, brown, circular or elliptical. Found free among egg mass of host or in a filiform, ornamented mucus sheath. Irregular lapilli on the mucus sheath (Figure 3); lapilli with concentric lines (Figure 4A). Anterior (Figure 4B) and posterior end of body rounded. Dimensions of relaxed worms were 7.0+ 4.0 mm (3.0-11.0 mm; w=2) long and 405+ 62 urn (230-500 urn; «=4) wide. Eye 14+ 2 urn (10-18 um; w=4) long and 9 + 1 um (8-12um; w=4) wide. Distance between eyes 131+ 14um (102-160um; «=4). Distance from eyes to tip of head 110+18 urn (70-150 um; w=4). Brain 99 + 6 urn (88- 110 um; «=3) long and 64 + 4 um (58-72 um; w=3) wide. Distance from brain to tip of head 150+ 23 urn (100-210um; w=4). Proboscis bent on itself. Anterior proboscis chamber could not be distinguished. Diaphragm 51+2 um (48-58 um; w=4) long and 56+ 3urn (50-62um; w=4) wide. Single stylet on basis 9+lum (8-10um; «=4) long. Stylet basis 22+1 um (20-25um; w=4) long and 8+1 um (8-10urn; w=4) wide (Figure4C). Stylet:basis ratio 0.416 (0.375-0.444; w=4). Distance from central stylet to tip of head 208+ 28 urn (170-290um; «=4). Accessory stylets absent. Proboscis bulb 924 C. Santos et al.

Figure 4. Carcinonemertes sebastianensis n. sp. (A) Detail of lapilli of adult mucus sheath. (B) Rounded anterior end of female, showing the great distance from first gonad to tip of head. (C) Stylet and basis (arrow) in a female worm. (D) Anterior end of male. (E) Posterior end of male. Abbreviations: b, proboscis bulb; d, diaphragm; g, gonad.

34 + 4(j.m (28-42 \im; w=4) long and 38 + 4|im (30-42 |jm; n=4) wide. Posterior proboscis could not be visualized. Ovaries arranged in one row on each side of the intestinal diverticula. Distance from first gonad to tip of head 1700+ 167 \im (1250— 2000pin; w=4) (Figure4B).

Male. Body color translucent white; gut yellow; gonads translucent white. Two eyes, brown, circular or elliptical; elliptical eyes the most common shape. Found free among New Carcinonemertes from Brazil 925

Figure 5. Carcinonemertes sebastianensis n. sp. (A) Egg strand (arrow) on Menippe nodifrons eggs. (B) Juvenile worms encysted (arrows) on the axis of the pleopod of Menippe nodifrons. egg mass of host or in a filiform, ornamented mucus sheath. Irregular lapilli on mucus sheath (Figure 3); lapilli with concentric lines (Figure 4A). Anterior and posterior end of body rounded (Figure4D, E). Dimensions of relaxed worms 6.2 + 1.4mm (2.0— 11.0mm; n=6) long and 362 + 28urn (280-480um; n=6) wide. Eye 15 + 1 urn (12- 18 um; M=6) long and 10+1 |im (5-12 um; w=6) wide. Distance between eyes 110 + 5 um (90-120 um; n=6). Distance from eyes to tip of head 112+Hum (80- 150 um; w=6). Brain 101 + 10um (75-125 um; w=5) long and 52 + 3 um (42-62 um; M=5) wide. Distance from brain to tip of head 150+ 13um (112-200 um; n=6). Proboscis bent in on itself. Anterior proboscis chamber could not be distinguished. Diaphragm 46 + 2um (40-50 um; w=6) long and 48+1 um (45-50 um; «=6) wide. Single stylet on basis ll+0um (10-12um; w=6) long. Stylet basis 25 + lum (22- 30um; M=6) long and 10 + 0um (8-1 Oum; n=6) wide (Figure4C). Stylet:basis ratio 0.431 (0.333-0.556; n=6). Distance from central stylet to tip of head 206 + 24um (150-280um; w=5). Accessory stylets absent. Proboscis bulb 30 + 2um (25-38um; M=6) long and 36 + 2|j.m (32-40\im; n=6) wide. Posterior proboscis 128+18um (100- 160 |im; %=3) long and 43 + 9 |rm (25-55 (J.m; w=3) wide. Seminal vesicle not visible under stereomicroscope, but could be seen under compound microscope in some specimens.

Egg. Color white. Egg strings (Figure 5A) found in a firm and transparent sheath. Developing eggs 86 + 2 um in diameter (75-100 um; n= 12). Egg strings 232 + 14 um (180- 260 um; n=5) wide. Number of eggs per egg string 386 + 66 (320-451; n=2). Number of rows of eggs per egg string 3 + 0 (n=5).

Larva. Body ciliated with anterior and posterior ciliary tufts. Body shape ovoid. Two eyes. Body length 92 um (w=l). Body width 62 um (w=l).

Infestation site Adult worms were found in the host's egg mass (ovigerous females with eggs in initial, intermediate and final stages of development). All immature worms were found encysted in a mucus sheath without ornamentation in the following sites: on the ventral side of the abdomen, on the setae that follow the border of the abdomen, on the central axis of 926 C. Santos et al. pleopods (Figure 5B), and on the setae of pleopods of non-ovigerous adult female crabs; on the ventral side of the abdomen, on the central axis of pleopods, and on the setae of pleopods of ovigerous females; at the ventral side of the abdomen, on the central axis of pleopods, and on the setae of pleopods of post-ovigerous females. No immature worms were found on male crabs.

Discussion The three species described here are gonochoric, do not present accessory stylets and, thereby, conform to the diagnosis of Carcinonemertidae provided by Humes (1942) and modified by Shields et al. (1989) and to the diagnosis of Carcinonemertes provided by Coe (1902). The genus Ovicides is characterized by being hermaphroditic and by having two accessory stylets (Shields 2001). Carcinonemertes divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. can be distinguished from each other by color of the body, shape of the posterior region of body, eye length, and by distance between the first ovary and tip of head (Table I). Carcinonemertes pinnotheridophila is found on the floor and wall of branchial chambers as well as on eggs of its hosts (McDermott and Gibson 1993). Juvenile worms of C. c. carcinophila, C. c. imminuta and C. mitsukurii are found between the gills of their hosts (Humes 1942; Shields 1992; Santos and Bueno 2001; Table II). None of the three species described here was found in the branchial chamber or between the gills of their hosts. The three species described here also can be distinguished from C. pinnotheridophila by the presence of eyes in the adult worms, shape of the egg mucus sheath and by the number of rows of ovaries on each side of the intestine (McDermott and Gibson 1993) (Table II). Carcinonemertes coei also has two rows of ovaries on each side of the intestine (Humes 1942; Table III). Carcinonemertes divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. can be distinguished from C. c. carcinophila by body length (Humes 1942), and from C. c. imminuta by body length and by eye width (Humes 1942; Table II). Adult worms of C. errans do not have a mucus sheath (Wickham 1978), whereas adult worms of C. regicides have a mucus sheath with no lapilli (Shields et al. 1989). The adult worms of the three species described here have a mucus sheath with lapilli, which are, according to Humes (1942), irregularly shaped, raised concretions, present on the surface of the sheath. In addition, C. errans can be distinguished from C. divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. by diaphragm length and by stylet basis length (personal

Table I. Morphological and ecological differences that distinguish the species Carcinonemertes divae n. sp., Carcinonemertes caissarum n. sp. and Carcinonemertes sebastianensis n. sp. (measurements in |j.m).

Characters C. divae n. sp. C. caissarum n. sp. C. sebastianensis n. sp. Body color White to orange White to cream, red spot White (male) Shape of posterior end of body Pointed Truncated on males Rounded Eye length 20-48 8-15 10-18 Distance 1 st ovary-tip of head 348-540 430-900 1250-2000 Infestation site e, ab, per, pi e, ab, gon, pi, sepl e, ab , seab, axpl, sepl

Table II. Morphological and ecological differences that distinguish the species Carcinonemertes carcinophila carcinophila, Carcinonemertes carcinophila imminuta, Carcinonemertes mitsukurii and Carcinonemertes pinnotheridophila from the species described here (body length in mm; other measurements in |j.m).

C. C.c. C.c. C. pinnotherido- C. divae C. caissarum n. C. sebastianensis n. Characters carcinophila imminuta mitsukurii phila n. sp. sp. sp. Body length 20.0-70.0 4.0-15.0 1.0-16.0 0.9-15.0 1.3-4.3 1.2-11.0 2.0-11.0 Eye width ? 20-70 ? eyeless 10-25 5-12 5-12 Number of 1112 1 1 1 rows of ovaries Shape of egg Filiform Filiform Filiform Oval Filiform Filiform Filiform mucus sheath Infestation site e, gl e, gl e, gl e, be e, ab, per, e, ab, gon, pi, e, ab, seab, axpl, pi sepl sepl

Abbreviations for infestation site: ab, abdomen; axpl, axis of pleopod; be, branchial chamber; e, eggs; gl, gill lamellae; gon, gonopores; per, pereiopods; pi, pleopods; seab, setae of abdomen; sepl, setae of pleopods. observation; Table III). The stylet basis in C. regicides is 38-44 \im long (Shields et al. 1989; Table III). Carcinonemertes australiensis can be distinguished from C. divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. by stylet basis length, stylet basis width and by stylet length (Campbell et al. 1989; Table III). The stylet basis is 10-12 |im wide (Gibson and Jones 1990) in C. humesi and 14|im (Shields and Kuris 1990) in C. wickhami. The latter also differs from C. divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. in diaphragm length, stylet basis length, stylet length and in stylet:basis ratio (Shields and Kuris 1990; Table IV). Stylet:basis ratio in C. humesi varies from 0.219 to 0.267 (Gibson and Jones 1990). Carcinonemertes divae n. sp., C. caissarum n. sp. and C. sebastianensis n. sp. can be distinguished from C. epialti by diaphragm width (personal observation; Table IV). Carcinonemertes epialti differs from C. divae new species in the alignment of proboscis and in

Table III. Morphological and ecological differences that distinguish the species Carcinonemertes coei, Carcinonemertes errans, Carcinonemertes regicides and Carcinonemertes australiensis from the species described here (measurements in Jim).

C. C. C. divae n. C. caissarum n.. C. sebastianensis Characters C. coei C. errans regicides australiensis sp. sp. n. sp. Presence of Present Absent Present ? Present Present Present mucus sheath Mucus sheath y N/A No lapilli ? Ornamented Ornamented Ornamented ornamentation Diaphragm y 62-125 64-96 75 42-80 30-60 40-58 length Stylet basis 21-23 30-40 38-44 40 22-30 18-25 20-30 length Stylet basis 6-8 10-13 9-17 15-16 5-10 5-8 8-10 width Stylet length 8-9 12 (no 14-19 15-18 8-12 5-10 8-12 variation) Number of rows 2 1 1 1 1 1 1 of ovaries 928 C. Santos et al.

Table IV. Morphological and ecological differences that distinguish the species Carcinonemertes wickhami, Carcinonemertes humesi and Carcinonemertes epialti from the species described here (measurements in |lm).

C. caissarum n. C. sebastianensis Characters C. wickhami C. humesi C. qpialti C. divae n. sp. sp. n. sp. Eye length 34- -39 ? 15- -28 20- -48 8- 15 10 -18 Distance eyes-tip of 126--196 ? 136- -200 100- -230 60- 155 70- 150 head Proboscis alignment ; > Curved Curved Straight line ? ? Diaphragm length 70- 112 J 50- -93 42- -80 30 -60 40- -58 Diaphragm width 56- -87 y 62- 112 38 -78 32 -55 45 -62 Stylet basis length 36--42 30-32 21- -23 22--30 18 -25 20-30 Stylet basis width 14 10-12 5- -8 5- 10 5 -8 8- 10 Stylet length 19- -20 7-8 10- -15 8- 12 5- 10 8- 12 Styleibasis ratio 0.476--0.528 0.219-0.267 0.333--0.500 0.250--0.500 0.250- -0.571 0.333 -0.556 the eye length (Coe 1902; TableIV). The eye is 8-15 \xm long in C. caissarum n. sp. and 10-18 |im long in C. sebastianensis n. sp. Carcinonemertes epialti also can be distinguished from C. caissarum n. sp. by stylet length (personal observation) and from C. sebastianensis new species by the distance between the eyes and tip of head (personal observation; Table IV). Juvenile worms of some nemerteans of the genus Carcinonemertes were previously found on the pleopods of their hosts (at the base or on the arthrodial membranes). A novelty regarding the infestation site by nemerteans of the genus Carcinonemertes is registered in the present study for C. sebastianensis n. sp., whose juveniles can be found encysted on setae of pleopods (a site also used by juveniles of C. caissarum n. sp.) or on the axis of pleopods, or even on setae that follow the border of the abdomen of the hosts. In addition to the diagnostics mentioned above, the three new species differ from other carcinonemertids in that they infest, respectively, L. spinosa, H. pudibundus and M. nodifrons, all registered here for the first time as hosts for nemerteans of the genus Carcinonemertes. Specimens of H. epheliticus and M. mercenaria collected at Grand Isle, Louisiana, USA, were registered by Humes (1942) as hosts for C. c. imminuta. Humes (1942) found juvenile worms on gills of H. epheliticus, which makes us believe he could be right about the identification of these specimens of Carcinonemertes. Nevertheless, the following facts suggest that there is a chance the worms found in H. epheliticus and M. mercenaria were misidentified as C. c. imminuta: (1) only one worm was found on the gill of H. epheliticus from Louisiana (Humes 1942); this infestation could be casual, not a regular pattern; (2) an adult male of H. epheliticus was collected in Florida and none of the 90 juvenile worms infesting the crab was found on gills (personal observation); (3) Humes (1942) did not find any worms on the gills of M. mercenaria and stated that "in the non-portunid species infested at Grand Isle, the worms were all minute, though were sexually mature"; it seems that the worms found in H. epheliticus and in M. mercenaria by Humes (1942) were smaller than C. c. imminuta. There are some factors that can lead to misidentifications among the species belonging to the family Carcinonemertidae: (1) the small size of worms, (2) morphological simplification apparently resulting from their parasite-like life style, and (3) the consequent morphological ambiguity of structures and morphological similarity between species. For example, the structure referenced as anterior proboscis chamber in some publications (Shields et al. 1989; Shields and Kuris 1990) appears to us to be the proboscis diaphragm and that is how New Carcinonemertes from Brazil 929 we treat it. Similarly, the region designated as the stylet bulb, appears to include the diaphragm in a number of publications (Gibson 1972; Campbell et al. 1989; Gibson and Jones 1990; McDermott and Gibson 1993); we distinguish between the two. The stifling morphological homogeneity of Carcinonemertes species led us to search for other, relatively practical characters, such as distance from first gonad to tip of head, distance from brain to tip of head and distance from stylet to tip of head. Distance from first gonad to tip of head enabled us to distinguish among the three species described here. We believe that information, such as infestation site and infestation according to sex and maturity of host, may also provide valuable diagnostic and systematic data. With this information in hand from previously described species it may be possible to conduct phylogenetic analyses with useful results that will lead to understanding of the diversification and host relationships of this potentially damaging group of nemerteans.

Acknowledgments We are grateful to the staff of the Departamento de Zoologia of the Instituto de Biociencias and the Centro de Biologia Marinha, Universidade de Sao Paulo, especially to Dr Claudio G. Tiago for providing logistic and laboratory facilities and to Elso Alves da Silva for his assistance in the field. We are grateful to the staff of the Smithsonian Marine Station at Fort Pierce, Florida, for providing logistic and laboratory facilities, especially to Julie Piraino, for her assistance with the scanning electron microscope. We thank two reviewers for helpful comments that improved this manuscript. We are grateful to FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo) for providing a scholarship grant (process number 01/01797-2) to CS and to the Link Foundation for providing a 12-week graduate student fellowship to CS. This is contribution No. 660 from the Smithsonian Marine Station at Fort Pierce. The work was supported in part by travel funds from the Smithsonian Marine Science Network and JLN's participation was supported in part by NSF PEET grant DEB 9712463.

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